Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a

Oct 5, 2019Applied microbiology and biotechnology

Using genome editing and gene suppression together to change metabolism in Corynebacterium glutamicum with active Cas12a

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Abstract

The application of the RE-CRISPR system increased cysteine production by 3.7-fold and serine production by 2.5-fold in C. glutamicum.

A dual-functional CRISPR system was developed for genome editing and transcriptional repression.
Gene deletion was successfully achieved using a catalytically active version of Cas12a under a constant promoter.
Transcriptional repression was effective with engineered guide RNA sequences, achieving similar efficiency to traditional methods.
Simultaneous genome editing and transcriptional repression were accomplished by using a single plasmid for crRNA and Cas12a.
This approach expands the use of CRISPR technology for metabolic engineering in C. glutamicum.

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Corynebacterium glutamicum is a versatile workhorse for producing industrially important commodities. The design of an optimal strain often requires the manipulation of metabolic and regulatory genes to different levels, such as overexpression, downregulation, and deletion. Unfortunately, few tools to achieve multiple functions simultaneously have been reported. Here, a dual-functional clustered regularly interspaced short palindromic repeats (CRISPR) (RE-CRISPR) system that combined genome editing and transcriptional repression was designed using a catalytically active Cas12a (a.k.a. Cpf1) in C. glutamicum. Firstly, gene deletion was achieved using Cas12a under a constitutive promoter. Then, via engineering of the guide RNA sequences, transcriptional repression was successfully achieved using a catalytically active Cas12a with crRNAs containing 15 or 16 bp spacer sequences, whose gene repression efficiency was comparable to that of the canonical system (deactivated Cas12a with full-length crRNAs). Finally, RE-CRISPR was developed to achieve genome editing and transcriptional repression simultaneously by transforming a single crRNA plasmid and Cas12a plasmid. The application of RE-CRISPR was demonstrated to increase the production of cysteine and serine for ~ 3.7-fold and 2.5-fold, respectively, in a single step. This study expands the application of CRISPR/Cas12a-based genome engineering and provides a powerful synthetic biology tool for multiplex metabolic engineering of C. glutamicum.

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Combined genome editing and transcriptional repression for metabolic pathway engineering in Corynebacterium glutamicum using a catalytically active Cas12a

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